RESUMO
After rapid multiple extractions of mouse plasma virus with ether, the aqueous solution contained viral nucleoids that were infectious when inoculated intracranially into newborn BALB/c mice. The infectivity associated with the ether extract was not neutralized by the specific antibody prepared against the whole virus. No intact virus has been seen in these preparations. Treatment with ether completely removed the virus envelope from the particle and produced an apparently homogeneous preparation of viral nucleoids. After the extractions with ether, leukemogenic activity was inactivated by exposure to ribonuclease. The leukemogenic activity of the many-passaged Rauscher virus that has been propagated in tissue culture and that has low infectivity was also retained, and, in two experiments in which material was inoculated intracranially into mice, this activity appeared to have been enhanced by multiple extractions with ether.
Assuntos
Vírus Rauscher/patogenicidade , Animais , Animais Recém-Nascidos , Técnicas de Cultura , Éteres , Camundongos , Microscopia Eletrônica , Testes de Neutralização , Vírus Rauscher/imunologia , Ribonucleases , VirulênciaRESUMO
Infecting mouse bone marrow cells (JLS-V9) with cell-free Rauscher leukemia virus demonstrated that monolayers of JLS-V9 cells were susceptible to infection with both cell-free plasma and tissue-culture-propagated Rauscher virus. The infected cultures maintained the continuous release of the virus. Cells and concentrated supernatant fluids from infected cells were periodically examined under the electron microscope by thin sectioning and negative staining techniques after inoculation with either mouse plasma or tissue-culture-propagated virus. After adsorption the virus underwent a prolonged eclipse or latent period. Virus budding was initially detected about 10 days after primary infection. These observations were further confirmed by the simultaneous appearance of extracellular virus. The budding process was followed by accelerated virus multiplication which reached maximum production 2-3 weeks later. Before the onset of budding no significant morphologic changes were seen in the infected cells.
Assuntos
Células da Medula Óssea/virologia , Microscopia Eletrônica , Vírus Rauscher , Animais , Células da Medula Óssea/ultraestrutura , Linhagem Celular/virologia , Camundongos , Plasma/virologia , Infecções por Retroviridae , Baço/citologia , Baço/virologia , Timo/citologia , Timo/virologia , Infecções Tumorais por VírusRESUMO
Simultaneous replication of murine mammary tumor virus (type B) and murine leukemia virus (type C) was demonstrated electron microscopically along continuous stretches of the plasma membrane of single cells in cultures ofthe Mm5mt/c1 cell line. Types B and C virus buds were discriminated in thin sections with the aid of a tannic acid fixative that revealed the type B surface spikes as a homogeneous band of intermediate density and constant width on the surfaces of some buds (type B), whereas others (type C) remained with relatively smooth envelopes. Both types B and C buds may contain morphologically identical horseshoe-shaped nucleoids. Therefore, their identify (type B or C) could be ascertained in thin sections only on the basis of recognition of surface spikes.
Assuntos
Vírus da Leucemia Murina/ultraestrutura , Vírus do Tumor Mamário do Camundongo/ultraestrutura , Replicação Viral , Taninos Hidrolisáveis , Corpos de Inclusão Viral , Microscopia EletrônicaAssuntos
Células da Medula Óssea , Medula Óssea , Vírus da Leucemia Murina de Moloney , Vírus Rauscher , Animais , Efeito Citopatogênico Viral , Camundongos , Microscopia Eletrônica , Vírus da Leucemia Murina de Moloney/imunologia , Testes de Neutralização , Vírus Rauscher/imunologia , Cultura de VírusAssuntos
Vacinas Bacterianas/normas , Cricetinae , Cobaias , Leptospira/imunologia , Animais , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/efeitos adversos , Vacinas Bacterianas/isolamento & purificação , Meios de Cultura , Estudos de Avaliação como Assunto , Injeções Intraperitoneais , Leptospira/crescimento & desenvolvimento , Leptospira interrogans/crescimento & desenvolvimento , Leptospira interrogans/imunologia , Leptospira interrogans serovar canicola/crescimento & desenvolvimento , Leptospira interrogans serovar canicola/imunologia , Leptospirose/imunologia , Leptospirose/veterinária , MasculinoAssuntos
Nefropatias/veterinária , Leptospira interrogans serovar canicola/imunologia , Leptospira interrogans/imunologia , Leptospirose/veterinária , Doenças dos Suínos/imunologia , Vacinação/veterinária , Animais , Vacinas Bacterianas/administração & dosagem , Bioensaio , Sangue/microbiologia , Cricetinae , Injeções Intraperitoneais , Injeções Subcutâneas , Nefropatias/imunologia , Nefropatias/microbiologia , Leptospira interrogans/isolamento & purificação , Leptospira interrogans serovar canicola/isolamento & purificação , Leptospirose/imunologia , Leptospirose/microbiologia , Suínos , Doenças dos Suínos/microbiologia , Urina/microbiologia , Doença de Weil/veterináriaAssuntos
Formação de Anticorpos , Leptospira interrogans serovar canicola/imunologia , Leptospira interrogans/imunologia , Suínos/imunologia , Testes de Aglutinação , Animais , Vacinas Bacterianas/administração & dosagem , Cricetinae , Imunização Passiva , Leptospirose/imunologia , Leptospirose/veterinária , Doenças dos Suínos/imunologia , VacinaçãoAssuntos
Vacinas Bacterianas/normas , Cricetinae , Cobaias , Leptospira/imunologia , Animais , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/efeitos adversos , Estudos de Avaliação como Assunto , Injeções Intraperitoneais , Leptospira interrogans/imunologia , Leptospira interrogans serovar canicola/imunologia , Leptospirose/imunologia , Leptospirose/veterinária , Masculino , Vacinação/veterináriaAssuntos
Doenças dos Bovinos/prevenção & controle , Nefropatias/veterinária , Leptospira interrogans serovar canicola/imunologia , Leptospira interrogans/imunologia , Leptospirose/veterinária , Vacinação/veterinária , Testes de Aglutinação , Animais , Vacinas Bacterianas/administração & dosagem , Sangue/microbiologia , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/patologia , Cricetinae , Imunização Passiva , Nefropatias/imunologia , Nefropatias/patologia , Nefropatias/prevenção & controle , Leptospira interrogans/isolamento & purificação , Leptospira interrogans serovar canicola/isolamento & purificação , Leptospirose/imunologia , Leptospirose/patologia , Leptospirose/prevenção & controle , Fígado/patologia , Suínos , Urina/microbiologiaRESUMO
JLS-V9, a mouse bone marrow cell line infected with Rauscher leukemia virus at high passage level, produced larger amounts of virus than the standard JLS-V10 cells. The enhanced virus production was attributed to the increased saturation density of JLS-V9 cells.
Assuntos
Vírus Rauscher/crescimento & desenvolvimento , Cultura de Vírus/métodos , Replicação Viral , Animais , Divisão Celular , Linhagem Celular , CamundongosRESUMO
Treatment of Rauscher murine leukemia virus lysates with the anionic detergent sodium dodecyl sulfate (SDS) at concentrations between 0.2 to 2.0% SDS per mg of viral protein greatly increased the anodal electrophoretic mobility of p30, the major internal polypeptide. SDS treatment did not reduce p30 antigenicity or cause nonspecific precipitation of normal serum proteins during subsequent immunoanalysis. The increased anodal electrophoretic mobility allowed assay of Rauscher murine leukemia virus p30 by Laurell rocket immunoelectrophoresis. An SDS-facilitated rocket immunoelectrophoresis assay is described that was highly reproducible (coefficient of variability, less than 3.0%) and capable of detecting 125 ng of viral protein. To our knowledge, this is the first report of a quantitative immunoelectrophoretic assay for an oncornavirus antigen. Since SDS binding is a general property of proteins, this method of noncovalently altering electrophoretic mobility appears to be applicable to other antigen-antibody systems.
Assuntos
Imunoeletroforese/métodos , Vírus Rauscher/análise , Proteínas Virais/análise , Carbamatos , Ponto Isoelétrico , Dodecilsulfato de Sódio , Proteínas Virais/imunologiaRESUMO
Rauscher murine leukemia virus was produced in roller-bottle cultures of chronically infected JLS-V9 cells. Virus from this culture fluid was concentrated and purified by two semi-isopycnic bandings in sucrose gradients. Virus material obtained from young, nonconfluent cultures (early-harvest virus) yielded products characteristically containing endogenous ribonucleic acid-dependent deoxyribonucleic acid polymerase with high specific activity (400 to 1,000 pmol of [3H]thymidine 5'-triphosphate incorporated per milligram of protein per hour). Fluids obtained from older confluent cultures (late-harvest virus) yielded products with endogenous ribonucleic acid-dependent deoxyribonucleic acid polymerase with little or no specific activity (200 pmol or less of [3H]thymidine 5'-triphosphate incorporated per milligram of protein per hour), but with higher virus particle counts and greater amounts of protein and gs antigen than the early-harvest products.
Assuntos
Vírus Rauscher/crescimento & desenvolvimento , Cultura de Vírus/métodos , Linhagem Celular , Sistema Livre de Células , DNA Polimerase Dirigida por RNA/metabolismo , Vírus Rauscher/imunologia , Vírus Rauscher/isolamento & purificação , Nucleotídeos de Timina/metabolismo , Proteínas Virais/biossíntese , Replicação ViralRESUMO
JLS-V9 mouse bone marrow cells were readily adapted to suspension culture, chronically infected with Rauscher leukemia virus (RLV), and subsequently grown in 7.5- and 14-liter New Brunswick fermentors. The suspension-type cell system can be modified to produce virus with clearly defined properties, such as high ribonucleic acid-dependent deoxyribonucleic acid polymerase (RDDP) activity, high particle count, and high infectious particle count. Biological and biophysical properties of suspension-produced RLV were not affected by concentration and purification employing continuous-flow and rate-zonal centrifugation procedures. The RDDP assay was standardized and showed a linear incorporation of (3)H-thymidine 5'-monophosphate ((3)H-TMP) up to 30 min. Further characterization indicated that a high percentage of (3)H-TMP incorporation was due to RDDP.
Assuntos
Células Cultivadas/microbiologia , Vírus Rauscher/crescimento & desenvolvimento , Animais , Células da Medula Óssea , Contagem de Células , Linhagem Celular , Sistema Livre de Células , Centrifugação Zonal , DNA Viral/biossíntese , Leucemia Experimental , Camundongos , Microscopia Eletrônica , Ácido Fosfotúngstico , DNA Polimerase Dirigida por RNA/metabolismo , Vírus Rauscher/enzimologia , Vírus Rauscher/isolamento & purificação , Vírus Rauscher/patogenicidade , Coloração e Rotulagem , Nucleotídeos de Timina/metabolismo , Trítio , Replicação ViralRESUMO
Large-scale production and concentration procedures have been standardized to study the biological properties of Rauscher leukemia virus produced from the high-passaged JLS-V9-H mouse bone marrow cell line. Virus produced early (days 4 to 6) in the harvest and refeed cycle contained higher levels of ribonucleic acid-directed deoxyribonucleic acid polymerase activity and was more infectious than Rauscher leukemia virus produced later (days 7 to 10) in the growth period. The peak of virus production as detected by physical assays (virus particle count, protein, and p30 antigen) was highest at day 6, whereas the optimum biological and ribonucleic acid-directed deoxyribonucleic acid polymerase activity occurred 24 h earlier. When product characterization values of each concentrate were adjusted to a specific activity (i.e., per milligram of protein) basis, virus particle counts averaged 4 x 10(11) through days 5 to 9, and the peak infectivity occurred at day 4, whereas ribonucleic acid-directed deoxyribonucleic acid polymerase activity was highest at day 4 (endogenous) and 5 (exogenous). Sodium dodecyl sulfate-polyacrylamide gel analysis revealed only slight differences in the polypeptide pattern of Rauscher leukemia virus harvested from cultures of varying age, although Rauscher leukemia virus produced between days 3 and 5 contained more glycoprotein than either earlier or later harvests.